Gene therapy provides exciting new approaches to treat numerous incurable neurodegenerative disorders such as Parkinson's disease, multiple sclerosis, or brain cancer. Unfortunately the immune response to therapeutic vectors remains a major obstacle to the clinical realization of gene therapy. If primed, the immune system eliminates expression of therapeutic transgenes from the brain, curtailing gene therapy's efficacy. We will investigate immune regulation of transgene expression mediated by clinically effective first generation adenoviral vectors, and novel high capacity 'gutless'adenoviral vectors in a clinically relevant model. In this model animals will be pre-immunized to adenovirus to mimic immune status in the majority of human patients that were exposed to adenovirus before receiving gene therapy. In this proposal we will test the hypothesis that the immune system eliminates expression of therapeutic transgenes from the brain, primarily through cytotoxic, and secondarily, through non-cytotoxic mechanisms. To address these issues, we developed a specific method to differentiate immune system-induced brain cell death from selective down-regulation of vector-mediated transgene expression. This method is based on transgenic mice containing a floxed beta-gal construct, and viral vectors expressing Cre under pancellular and cell type specific promoters. Infected cells thus express a gene marker from their genomes, expression of which will be regulated independently of expression from the viral vector's genome. Preliminary experiments using our new method demonstrate that the immune system utilizes both mechanisms, namely it can eliminate expression of therapeutic transgenes from the brain by [1] direct cytotoxicity of transduced cells;and, [2] functional inhibition of transgene expression. Herein we will test specific hypothesis concerning the immune mechanisms that eliminate brain transgene expression, in both males and females, and in two mouse strains;one that displays TH1 biased immune responses (C57BI/6), and another one that displays a TH2 bias (DBA/2J). As a result of this work we will make available enhanced and safer gene therapy approaches, and more efficient clinical treatment paradigms. The results from this proposal will have a direct impact on experimental and clinical gene therapy, as well as make major contributions to understanding how the immune system eliminates or regulates gene expression in virally infected brain cells.